以病毒誘導基因靜默技術探討PhLA基因在矮牽牛花型態之影響

Abstract

自然界中有形形色色的花，植物發展出此一多變多彩的誘人器官，不外乎是為了繁衍下一代。前人研究關於影響植物花器的基因中，CYC是一決定花朵兩側對稱的重要因子。CYC屬於TCP transcription factor，本論文研究以TCP domain序列對矮牽牛EST資料庫進行BLAST，得到兩段CYC-like的片段，利用virus-induced gene silencing (VIGS)技術觀察此二基因表現量的降低對花朵外觀的影響。結果發現花朵對稱性經VIGS處理後並無影響，然而以帶有其中一個基因片段的病毒感染之矮牽牛在花朵向外的開展程度中與控制組花朵有明顯的差異。將此一基因cDNA片段進行RACE PCR取得全長的轉譯區後，將其衍生之胺基酸序列進行序列比對，發現其為TCP transcription factor上屬於CIN subgroup之蛋白，與金魚草(Antirrihnum majas)的CINCINNATA、阿拉伯芥(Arabidopsis thaliana L. )的TCP4以及蕃茄(Solanum lycopersicon L.)中的LA親緣關係相近，其中SlLA可能為直向同源基因(ortholog)，因此命名此基因為PhLA。前人對AmCIN、AtTCP4和SlLA的研究顯示，在此三物種中這些基因的突變株，同時影響花和葉的型態，但在矮牽牛中PhLA表現量的降低僅在花的型態有顯著差異。本研究針對矮牽牛VIGS處理後觀察到的性狀為基礎，進行RT-PCR半定量、原位雜交和電子顯微鏡觀察細胞等技術分析PhLA在矮牽牛花朵的生長發育上所扮演的角色。目前研究結果顯示，當PhLA表現量下降時，花冠翼中脈部份的細胞由原本立體錐狀轉為較大且立體感下降的平坦細胞，與在VIGS處理植株花朵，花冠翼測量得到的較小最小寬、較大最大寬和較長的高有正向關係。此外，PhLA所屬的CIN-subgroup基因多有一miR319的辨識位置，前人研究在阿拉伯芥與蕃茄當中都證實miR319的表現會造成目標基因表現量的下降，進而對花朵或葉的生長有影響。矮牽牛PhLA中也在靠近3’端處定位出一miR319辦識位置，推測PhLA亦可受miR319調控，並且此一調控可能為決定花朵型態的重要因子。本研究利用VIGS技術，了解PhLA在演化中所扮演的角色，同時探討PhLA與miR319間的調控機制對花朵開展性的影響。

The plant develops various shapes of flowers in nature in order to attract pollinators for increasing the progenies of next generation. Asymmetry is one special trait for distinguishing different species of flowers. In previous research, CYC was discovered to be a major gene deciding the dorsal-ventral asymmetry of the flower in Antirrhinum. CYC is a TCP transcription factor and the BLAST result from petunia EST database identified two CYC-like DNA segments and applied to virus-induced gene silencing (VIGS) for petunia cultivar ‘Primtime Blue’. In the VIGS treated plants with reduced target gene expression based on the CYC-like segments, comparing to the control plant instead of showing different morphology of flower asymmetry, greater curvature of the petal lobe was observed. The full cDNA sequence of the silenced gene was obtained by RACE PCR and its derived amino acid sequence was used for alignment with other genes. The alignment result classified this gene into TCP gene family and the CIN subgroup, is closely related to its homologs CINCINNATA in snapdragon (Antirrihnum majas), TCP4 in Arabidopsis (Arabidopsis thaliana L. ) and an ortholog of LA in tomato (Solanum lycopersicon L.), thus named the gene as PhLA. Previous research about AmCIN, AtTCP4 and SlLA in either mutants or transformants showed defects in both leaves and flowers, however in petunia only exhibits abnormal growth in the flower when PhLA expression is down regulated. In this research, based on the phenotype observed in the VIGS treated plants, a number of techniques such as RC-PCR semi-quantification, in situ hybridization and scanning electronic microscopy (Hennig et al.) were used to analyze the role of PhLA in the development of petunia flowers. So far, it is indicated cells around the central vein area of petal lobes turned from conical shape to flatter and greater type which is parallel to the phenotype of petal lobes measured of greater maximum width, shorter minimum width and greater heights. Moreover, a miR319 recognition site near the 3’-UTR of PhLA was identified; previous research of miR319 in Arabidopsis and tomato verified that its expression affects the growth of both flowers and leaves by down regulating target genes. It is suspected that PhLA is regulated by miR319 and may be a deciding factor of flower morphology. In summary, it is expected to understand the evolutionary role of PhLA and the influence of regulatory mechanism of miR319 and PhLA to the flower curvature with the use of VIGS technique.